WO1996005213A1 - Oligomeres se fixant sur des sequences specifiques d'acides nucleiques et leur utilisation dans des strategies d'anti-sens - Google Patents

Oligomeres se fixant sur des sequences specifiques d'acides nucleiques et leur utilisation dans des strategies d'anti-sens Download PDF

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Publication number
WO1996005213A1
WO1996005213A1 PCT/EP1995/003248 EP9503248W WO9605213A1 WO 1996005213 A1 WO1996005213 A1 WO 1996005213A1 EP 9503248 W EP9503248 W EP 9503248W WO 9605213 A1 WO9605213 A1 WO 9605213A1
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WO
WIPO (PCT)
Prior art keywords
oligomers
formula
heterocyclic ring
mmol
sequence
Prior art date
Application number
PCT/EP1995/003248
Other languages
English (en)
Inventor
Piet André Maurits HERDEWIJN
Arthur Albert Edgard Van Aerschot
Original Assignee
Stichting Rega Vzw
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Stichting Rega Vzw filed Critical Stichting Rega Vzw
Priority to JP08507032A priority Critical patent/JP2000505778A/ja
Priority to EP95930468A priority patent/EP0777676A1/fr
Priority to NZ292140A priority patent/NZ292140A/en
Priority to AU33845/95A priority patent/AU3384595A/en
Publication of WO1996005213A1 publication Critical patent/WO1996005213A1/fr
Priority to FI970598A priority patent/FI970598A0/fi
Priority to MXPA/A/1997/001111A priority patent/MXPA97001111A/xx
Priority to NO970716A priority patent/NO970716L/no

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
    • C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to oligomers having nucleic acid binding properties, which oligomers completely or partially consist of 1,5-anhydrohexitol nucleoside analogues as monomeric units.
  • the invention further relates to the use of the oligomers in antisense techniques and to a method of preparing the oligomers.
  • Antisense techniques are based on the principle that the function of a coding sense strand of a DNA or RNA molecule may be blocked by a complementary antisense strand. Antisense techniques may be used for various applications, such as diagnosis, therapy, DNA modification and isolation etc..
  • the stability of the antisense strand itself besides the stability of the antisense strand itself, the stability of the duplex or triplex formed by the sense and antisense strands as well as the binding affinity of the antisense strand for the sense strand are of importance.
  • the sensitivity of the oligomer, the duplex or the triplex for degrading enzymes, such as nucleases, is a factor relevant for the effectivity.
  • Oligonucleotides are oligomers in which the monomers are nucleotides.
  • Nucleotides are phosphate esters of nucleosides, which are built of a purine or pyrimidine base and a sugar.
  • the backbone of each nucleotide consists of alternating sugars and phosphate groups.
  • the stability and binding affinity of the nucleotides may for example be influenced by modification of the base. Research in that direction (1-5) showed that such modifications only lead to less stable duplexes. Alterations in the backbone or the incorporation of new structures therein did lead to an increased nuclease stability but had only an adverse effect on their binding affinity for complementary strands. Modification of the sugars led to a merely limited increase in the affinity for the target molecule (6-8) . It is the object of the present invention to provide new oligomers, which have an improved stability and binding affinity as compared to the known oligomers.
  • oligomers consisting completely or partially of l,5-anhydro-2,3-dideoxy-D- arabino-hexitol nucleoside analogues, wherein the hexitol is coupled via its 2-position to the heterocyclic ring of a pyrimidine or purine base, are capable of binding to naturally occurring oligonucleotides.
  • the monomers of which the oligomers are at least partially composed are presented by the formula I:
  • B is a heterocyclic ring which is derived from a pyrimidine or purine base and, wherein 1 is an integer from 0 to 15, k and m each are integers from 1 to 15, but if k > 1, then m may be 0 and if m > 1, k may be 0; and, wherein X represents oxygen or sulfur.
  • All possible salts of the compound of formula II are included in the invention.
  • the monomers of formula I are the subject of European patent application No. 92201803.1.
  • the oligomers of formula II are novel compounds.
  • oligomers according to the invention consisting at least partially of pyranose nucleosides, have a high binding affinity is very surprising.
  • the study of oligonucleotides built up from monomeric pyranose nucleotides has been undertaken over the past years inter alia by the group of A. Eschenmoser et al..
  • Eschenmoser investigated nature's selection of furanoses as sugar building blocks for nucleic acids (9) .
  • pyranose-like oligonucleotide would be able to form stable duplexes with natural furanose-DNA (10, 11) .
  • a pyranose oligonucleotide has a free energy advantage over a furanose oligomer because of less entropy changes during duplex formation.
  • the pyranose-like oligonucleotides studied by the present inventors before were not able or not sufficiently able to bind to complementary strands of natural furanose-DNA.
  • pyranose-like oligonucleotides consisted of 2,3-dideoxy-B-D-erythro-hexopyranosyl nucleosides (formula V), 2,4-dideoxy- ⁇ -D-erythro- hexopyranosyl nucleosides (formula VI) and/or 3,4-dideoxy- ⁇ -D-erythro-hexapyranosyl nucleosides (formula VII) , respectively.
  • the compounds according to the invention are therefore oligomers of nucleoside analogues wherein a l,5-anhydro-2,3-dideoxy-D-hexitol is coupled via its
  • the oligomers consist of the above nucleoside analogues connected to each other as phosphate diesters or thiophosphate diesters.
  • the oligomers can be represented by the formula II wherein k, 1, m, B and X have the above stated designations.
  • the oligomers can be exclusively composed of the hexitol nucleoside analogues of the formula I (with 1 in formula II equalling zero) or can have natural 2 '-deoxynucleosides interspersed or at the end of the molecule (with 1 in formula II equalling one or greater) .
  • the hexitol has the (D)-configuration and the stereochemistry of the substituents is according to an arabino configuration.
  • group B When group B is derived from a pyrimidine base it can be either cytosine, 5-methyl cytosine, uracil or thymine. When B is derived from a purine base it can be an adenine, guanine, 2, 6-diaminopurine, hypoxanthine or xanthine ring, or a deaza derivative of one to these.
  • nucleoside analogues, monomer components of the present invention can be prepared in different ways and one of the preparation methods is subject of the European patent application no. 92.201803.1. These syntheses haven been described likewise in Verheggen et al. (12) . Assembly of the monomers into an oligomer follows the classical schemes and can be done either by standard phosphoramidite chemistry (compare ref. 13) or by H-phosphorate chemistry (compare ref. 14) . All procedures are conveniently carried out on an automated DNA synthesizer as for standard oligonucleotide synthesis. For these standard conditions reference is made to Methods in Molecular Biology (15) .
  • the preferred method is the phosphoramidite method making use of the phosphoramidites of the hexitol nucleoside analogues as the incoming building blocks for assembly in the "6'-direction" .
  • the phosphoramidites are represented by formula VIII wherein B * is a protected base moiety suitable for oligonucleotide synthesis (e.g. thymine, N 4 -benzoyl- cytosine, N 6 -benzoyladenine en N 2 -isobutyrylguanine, represented by the formula's IX, X, XI and XII, respectively) .
  • the products of formula VIII can be prepared according to standard procedures. Protection of the base moieties of cytosine, adenine or guanine is accomplished following a transient protection strategy for the hydroxyl moieties of the compounds of formula I (16) . Preferably, however, the base protection is carried out by acylation of the 4,6-benzylidene protected nucleoside analogues la-d, which are intermediates in the synthesis of the monomers of the above stated formula I.
  • the benzylidene moiety is removed with 80% acetic acid to obtain 3a-d.
  • the p-nitro-phenylethyl group can be removed with DBU.
  • the primary hydroxyl function of the 1,5-anhydro- hexitol analogues 3a-d can be protected with a dimethoxy- trityl group to yield 4a-d.
  • Conversion to the phosphor ⁇ amidite building blocks 5a-d suitable for incorporation into an oligonucleotide chain can be accomplished with 2-cyano- ethyl N,N-diisopropylchlorophosphoramidite.
  • Supports containing a 1,5-anhydrohexitol analogue can be prepared by succinylation of the compounds 4a-d yielding 6a-d, which can be coupled to the amino function of either long chain alkylamino controlled pore glass (CCAA-CPG) or a suitable amino functionalized polystyrene (e.g. Tentagel ⁇ -RAPP Polymere) making use of a carbodiimide, and yielding 7a-d (for functionalization of supports viz. ref. 17) After assembly, the obtained oligonucleotides are cleaved from the support and deprotected by ammonia treatment for 16 hours at 55°C.
  • CCAA-CPG long chain alkylamino controlled pore glass
  • a suitable amino functionalized polystyrene e.g. Tentagel ⁇ -RAPP Polymere
  • Purification of the obtained oligomers of the above stated formula II can be accomplished in several ways (18) .
  • the preferred method is purification by anion-exchange FPLC at a basic pH of 12 to disrupt all possible secondary structures (10) .
  • Desalting can be performed by simple gel filtration techniques followed by lyophilization. All acceptable salts can be prepared in conventional manner.
  • the oligomers display sequence- specific binding to natural oligonucleotides. They show stronger binding to a complementary natural oligodeoxy- nucleotide than the unmodified sequence and they are endowed with much higher biochemical stability. In this manner they can advantageously be used for antisense strategies which comprise diagnosis, hybridization, isolation of nucleic acids, site-specific DNA modification and therapeutics and all anti-sense strategies currently being pursued with natural oligodeoxynucleotides.
  • FABMS fast atom bombardment mass spectrometry
  • the unreacted sites on the surface of the support were capped using 1.5 ml of 1-methylimidazole in THF (Applied Biosystems) and 1.5 ml of acetic anhydride- lutidine-THF 1:1:8 (Applied Biosystems). After shaking for 4 hours at room temperature, the solid support was filtered off, washed with CH 2 C1 2 and dried under vacuum. Colorimetric dimethoxytrityl analysis indicated a loading of 18.5 ⁇ mol/g for 7a and 21.5 ⁇ mol/g for 7b.
  • Oligonucleotide synthesis was performed on an ABI 381A DNA synthesizer (Applied Biosystems) using the phosphoramidite method (end dimethoxytrityl off) .
  • the obtained sequences were deprotected and cleaved from the solid support by treatment with concentrated ammonia (55 ⁇ C, 16 hours) .
  • the low pressure liquid chromatography system consisted of a Merck-Hitachi L6200 A Intelligent Pump, a Mono Q® HR 10/10 column (Pharmacia) , an Uvicord SJI 2138 UV detector (Pharmacia-LKB) and a recorder.
  • the product contai ⁇ ning fraction was desalted on a NAP-10® column and lyophili- zed.
  • oligoA * and oligoT * both show an ordered structure but, in contrast to the results at high salt concentration, (results not shown) polyT * does not show the same tendency for ho oduplex formation. This is demonstrated by the more or less linear increase of the UV absorption with temperature, both for oligoA * and oligoT * .
  • An equimolar mixture of oligoT * and oligodeoxyadenylate shows a melting temperature of 45°C with a hypochromicity of 49% when measured at 284 nm. It is known that, by changing salt concentration, structural transition occurs in DNA and this is here clearly the case.
  • the oligoT * oligodeoxyadenylate association is favored at lower salt concentration while the formation of oligoT * homoduplexes is favored at high salt concentrations.
  • the thermal behavior of the complex at 260 nm indicates that the oligoT * :oligodeoxy-adenylate association is not a classical helix-coil transition.
  • the hypochromicity first decreases, showing a minimum at 46°C (the melting point observed at 484 nm) and then increases.
  • Fully modified mixed sequences two hexa ers and a dodecamer
  • containing the adenine (A * ) and guanine (G * ) nucleoside analogues have been evaluated likewise. Table 3
  • Duplexes were formed with the complementary sequences 5'-TCTCCT(20) for 16 and 17, and 5'-TCTCTC(21) for 18 and 19 respectively.

Abstract

L'invention concerne des oligomères constitués en partie ou en totalité de nucléosides du type 1,5-anhydrohexitol représentés par la formule générale (I). Dans cette formule, B est un groupe hétérocyclique dérivé d'une base pyrimidique ou purique, comme par exemple la cytosine, la 5-méthylcytosine, l'uracile et la thymine, ou leurs dérivés désaza, ou encore comme par exemple l'adénine, la guanine, la 2,6-diaminopurine, l'hypoxanthine et la xanthine ou leurs dérivés désaza.
PCT/EP1995/003248 1994-08-17 1995-08-14 Oligomeres se fixant sur des sequences specifiques d'acides nucleiques et leur utilisation dans des strategies d'anti-sens WO1996005213A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
JP08507032A JP2000505778A (ja) 1994-08-17 1995-08-14 核酸に関する配列特異的結合性オリゴマーおよびアンチセンスストラテジーにおけるそれらの用途
EP95930468A EP0777676A1 (fr) 1994-08-17 1995-08-14 Oligomeres se fixant sur des sequences specifiques d'acides nucleiques et leur utilisation dans des strategies d'anti-sens
NZ292140A NZ292140A (en) 1994-08-17 1995-08-14 Oligomers consisting of 1,5-anhydrohexitol nucleosides, antisence techniques.
AU33845/95A AU3384595A (en) 1994-08-17 1995-08-14 Sequence-specific binding oligomers for nucleic acids and their use in antisense strategies
FI970598A FI970598A0 (fi) 1994-08-17 1997-02-12 Nukeliinihappoihin sekvenssipesifisesti sitoutuvat oligomeerit ja niiden käyttö antisense-menetelmissä
MXPA/A/1997/001111A MXPA97001111A (en) 1994-08-17 1997-02-12 Specific oligomeros of sequence union nucleic paraacidos and its use in antisent strategies
NO970716A NO970716L (no) 1994-08-17 1997-02-17 Sekvens-spesifikke bindingsoligomerer for nukleinsyrer og deres anvendelse i antisense-strategier

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP94202342 1994-08-17
NL94202342.5 1994-08-17
US49515295A 1995-06-27 1995-06-27
US08/495,152 1995-06-27

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WO1996005213A1 true WO1996005213A1 (fr) 1996-02-22

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EP (1) EP0777676A1 (fr)
JP (1) JP2000505778A (fr)
CN (1) CN1158618A (fr)
AU (1) AU3384595A (fr)
CA (1) CA2196306A1 (fr)
FI (1) FI970598A0 (fr)
HU (1) HUT77509A (fr)
NO (1) NO970716L (fr)
NZ (1) NZ292140A (fr)
WO (1) WO1996005213A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030064A1 (fr) * 1996-02-16 1997-08-21 Stichting Rega Vzw Oligonucleotides contenant de l'hexitol et leur utilisation dans des strategies antisens
EP1431298A1 (fr) * 2002-12-20 2004-06-23 Roche Diagnostics GmbH Dérivés de manitol et glucitol
EP1431297A1 (fr) * 2002-12-20 2004-06-23 Boehringer Mannheim Gmbh Dérivés de mannitol et glucitol
EP1466919A1 (fr) * 2003-04-05 2004-10-13 Roche Diagnostics GmbH Analogues nucleotidiques hexacycliques
WO2006047842A2 (fr) * 2004-11-08 2006-05-11 K.U. Leuven Research And Development Nucleosides modifies pour interference arn
US7205106B1 (en) 2001-07-20 2007-04-17 Roche Molecular Systems, Inc. Association of polymorphisms in IL4-related genes with autoimmune disease
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
US7560231B2 (en) 2002-12-20 2009-07-14 Roche Molecular Systems, Inc. Mannitol and glucitol derivatives
WO2009100320A2 (fr) * 2008-02-07 2009-08-13 Isis Pharmaceuticals, Inc. Analogues d’acides nucléiques de cyclohexitol bicycliques
US8026068B2 (en) 2002-01-08 2011-09-27 Roche Molecular Systems, Inc. Use of silica material in an amplification reaction

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025565A1 (fr) * 1992-06-18 1993-12-23 Stichting Rega Vzw Analogues de nucleosides a base de 1,5-anhydrohexitol et leur utilisation pharmaceutique
US5314893A (en) * 1993-01-25 1994-05-24 Bristol-Myers Squibb Co. Antiviral tetrahydropyrans

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025565A1 (fr) * 1992-06-18 1993-12-23 Stichting Rega Vzw Analogues de nucleosides a base de 1,5-anhydrohexitol et leur utilisation pharmaceutique
US5314893A (en) * 1993-01-25 1994-05-24 Bristol-Myers Squibb Co. Antiviral tetrahydropyrans

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AUGUSTYNS K. ET AL.: "HYBRIDIZATION SPECIFICITY, ENZYMATIC ACTIVITY AND BIOLOGICAL (HA-RAS) ACTIVITY OF OLIGONUCLEOTIDES CONTAINING 2,4-DIDEOXY-BETA-D-ERYTHRO-HEXOPYRANOSYL NUCLEOSIDES", NUCLEIC ACIDS RESEARCH, vol. 21, no. 20, OXFORD GB, pages 4670 - 4676 *
AUGUSTYNS K. ET AL.: "INCORPORATION OF HEXOSE NUCLEOSIDE ANALOGUES INTO OLIGONUCLEOTIDES: SYNTHESIS AND BASE-PAIRING PROPERTIES AND ENZYMATIC STABILITY", NUCLEIC ACIDS RESEARCH, vol. 20, no. 18, OXFORD GB, pages 4711 - 4716 *
VAN AERSCHOT A.: "1,5-ANHYDROHEXITOL NUCLEIC ACIDS, A NEW PROMISING ANTISENSE CONSTRUCT", ANGEWANDTE CHEMIE INTERNATIONAL EDITION., vol. 34, no. 12, 7 July 1995 (1995-07-07), WEINHEIM DE, pages 1338 - 1339 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997030064A1 (fr) * 1996-02-16 1997-08-21 Stichting Rega Vzw Oligonucleotides contenant de l'hexitol et leur utilisation dans des strategies antisens
US7205106B1 (en) 2001-07-20 2007-04-17 Roche Molecular Systems, Inc. Association of polymorphisms in IL4-related genes with autoimmune disease
US8026068B2 (en) 2002-01-08 2011-09-27 Roche Molecular Systems, Inc. Use of silica material in an amplification reaction
EP1431298A1 (fr) * 2002-12-20 2004-06-23 Roche Diagnostics GmbH Dérivés de manitol et glucitol
EP1431297A1 (fr) * 2002-12-20 2004-06-23 Boehringer Mannheim Gmbh Dérivés de mannitol et glucitol
US7560231B2 (en) 2002-12-20 2009-07-14 Roche Molecular Systems, Inc. Mannitol and glucitol derivatives
EP1466919A1 (fr) * 2003-04-05 2004-10-13 Roche Diagnostics GmbH Analogues nucleotidiques hexacycliques
US7276592B2 (en) 2003-04-05 2007-10-02 Roche Diagnostics Operations, Inc. Nucleotide analogs with six-membered rings
WO2006047842A2 (fr) * 2004-11-08 2006-05-11 K.U. Leuven Research And Development Nucleosides modifies pour interference arn
WO2006047842A3 (fr) * 2004-11-08 2006-09-28 Leuven K U Res & Dev Nucleosides modifies pour interference arn
WO2009100320A2 (fr) * 2008-02-07 2009-08-13 Isis Pharmaceuticals, Inc. Analogues d’acides nucléiques de cyclohexitol bicycliques
WO2009100320A3 (fr) * 2008-02-07 2009-11-05 Isis Pharmaceuticals, Inc. Analogues d’acides nucléiques de cyclohexitol bicycliques

Also Published As

Publication number Publication date
FI970598A (fi) 1997-02-12
JP2000505778A (ja) 2000-05-16
HUT77509A (hu) 1998-05-28
EP0777676A1 (fr) 1997-06-11
CA2196306A1 (fr) 1996-02-22
AU3384595A (en) 1996-03-07
CN1158618A (zh) 1997-09-03
FI970598A0 (fi) 1997-02-12
NZ292140A (en) 1998-02-26
NO970716L (no) 1997-02-17
MX9701111A (es) 1998-03-31

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